The Effect of Chronic Contractile Activity and Retinoic Acid on Mitochondrial Turnover in C2C12 Myotubes

Abstract

Mitochondrial turnover is necessary for the optimization and maintenance of a healthy pool of functional organelles, able to meet the metabolic needs of the muscle cell. Exercise is known to improve mitochondrial turnover by activating both mitochondrial biogenesis and mitochondrial‐specific autophagy, known as mitophagy. Moreover, research in adipocytes and liver tissue has shown the potential of the dietary supplement retinoic acid to activate mitochondrial biogenesis as well. Retinoids have also been shown to be effective in activating autophagy in cancer cells. However, the combined effect of retinoic acid isomers and exercise in facilitating mitochondrial turnover has not been described. The purpose of this project was to evaluate the effect of retinoic acid (RA) isomers combined with exercise on muscle mitochondrial turnover. Mouse C2C12 myoblasts were plated on 6‐well plates and allowed to differentiate into myotubes. Myotubes were then treated with either vehicle (DMSO), or the retinoic acid (RA) isomers 9‐cis RA or All‐trans RA (ATRA) in the absence or presence of electrical stimulation to elicit Chronic Contractile Activity (CCA; 4days, 3hrs/day). Sample collection took place 21h after the fourth day. Mitochondrial biogenesis was assessed by examining the protein content of PGC‐1α, as well as nuclear and mitochondrially‐encoded mitochondrial proteins. CCA led to a 3‐fold increase in COX subunit IV expression, as well as a 2‐fold increase in PGC‐1α. The mtDNA‐encoded COX subunit I was modestly changed by CCA, but increased 2‐fold in response to 9cis‐RA or ATRA treatment. These RA isomers also increased PGC‐1α by 3‐fold, but had no significant impact on COX IV expression. When RA isomers were combined with CCA, no additive or synergistic effects were observed. To evaluate mitochondrial degradation, we examined several lysosomal and mitophagy proteins. While autophagy proteins Beclin and p62 showed no changes with CCA or RA treatment, the LC3II to LC3I ratio was reduced 2‐fold with CCA, suggesting an enhanced clearance of autophagosomes. RA isomers had no effect on this ratio. The lysosomal fusion protein Lamp1 was increased 1.8‐fold by ATRA, but not further by CCA. Our data suggest that while RA isomers and CCA both promote mitochondrial biogenesis, RA appears to target mitochondrially‐encoded proteins, while CCA appears to have greater effects on the expression of target nuclear‐encoded proteins. CCA also may serve as a stimulus for the clearance of autophagosomes containing damaged cargo. The lack of synergy among these pathways suggests that they operate independently to stimulate mitochondrial turnover.Support or Funding InformationSupported by NSERC, CanadaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.